Study On Chemoenzymatic Synthesis Of Unmodified Heparin Oligosaccharides And Preliminary Anticoagulant Activity

Heparin is a natural anticoagulant that has in vitro and in vivo effects.It is mainly used clinically for thromboembolic diseases,myocardial infarction,cardiovascular surgery,cardiac catheterization,extracorporeal circulation,hemodialysis,etc.With the advancement of pharmacology and clinical medicine,the application of heparin continues to be expanded.From the structural point of view,it is composed of repeating disaccharide units of uronic acid(L-iduronic acid or D-glucuronic acid)residue 1,4-linked to α-D-glucosamine residue.The heterogeneous polyanionic polysaccharides have different chain lengths and belong to highly sulfated glycosaminoglycans.Heparin exerts its anticoagulant activity depending on the antithrombin Ⅲ(ATⅢ)-binding pentasaccharide sequences that account for about 1/3 of the whole heparin chain.Natural heparin is limited by its production depending on a single animal species,and has potential safety issues related to animal-derived raw material.For this reason,chemical synthesis is used in the research and development of heparin,which led to success of fondaparinux sodium as a new anticoagulant in 2001.Nevertheless,fondaparinux is synthesized using not less than 50 steps,with a final yield of no more than 0.5%,resulting in extremely high production costs.Chemoenzymatic strategy has developed in recent years as a new method of heparin oligosaccharide synthesis.The method is based on multi-enzyme catalytic synthesis,supplemented by chemical synthesis.Compared with simply chemical synthesis,chemoenzymatic synthesis has the advantages of higher regioselectivity,less synthesis steps and higher synthesis yield,and mild reaction conditions.Therefore,based on the chemoenzymatic synthesis technology previously established at our laboratory,the current study was conducted by introducing more chemical steps and heparinase III cleavage to expand the chemoenzymatic synthesis strategy for the establishment of a chemoenzymatic synthesis route of unmodified heparin.And this strategy was used for synthesis of a natural heparin oligosaccharide containing AT-binding sequence,followed by characterization of its structure and determining its preliminary anticoagulant activity.The main results and conclusions of the study are as follows:1.Chemoenzymatic synthesis of unmodified heparin disaccharide receptorsThe classical chemoenzymatic synthesis is initiated from the p-nitrophenyl(PNP)-tagged glucuronic acid derivative(GlcA-PNP)as the starting monosaccharide receptor to give the resulting products of heparin oligomers carrying PNP at the reducing end that pose safety risks.In order to realize the chemoenzymatic synthesis of " unmodified " heparin oligosaccharides,this dissertation designed a disaccharide(GlcA-GlcNR,where R=Cbz or PNZ)that carries a chromophoric N-protecting group and is easy to be removed.The disaccharide is suitable as the starting receptor for sugar chain elongation.Combing the specific cleavage of heparinase with enzymatic sugar chain elongation led to efficiently synthesis of two disaccharide receptors 2mer-2/3 with 5 steps,88.4%or 81.2%yield.The other two disaccharides 2mer-4/5 were synthesized with 6 steps,44%the yield.2.Chemoenzymatic synthesis of unmodified heparin oligosaccharidesGlcA-GlcNPNZ(or GlcA-PNP)used as the starting material was elongated using enzymatic steps.The resulting backbone was modified by chemoenzymatic N-sulfation,C5-isomerization and 2-O-sulfation,supplemented by mild chemical modification and heparinase-III cleavage to give heparin oligosaccharides with different size and modification patterns,and the purity of each heparin oligosaccharide was>95%.Each heparin oligosaccharide was determined for molecular weight by ESI-MS and for structural characterization(if necessary)by NMR.Subsequent studies have shown as follows.Firstly,GlcNPNZ residues in oligomers can efficiently remove the PNZ group by palladium-carbon reduction.Secondly,GlcNH2 residues at the reducing end of oligosaccharides were not be enzymatically N-sulfated.Thirdly,GlcA residues adjacent to the non-reducing end were not converted into IdoA2S residues by C5-epimerase and 2-OST enzymes in one step,thus a heparin pentasaccharide with IdoA2S-GlcNS at the reducing end and>95%purity was obtained using 9 reaction steps,7 times of purification and specific cleavage of heparinase III.Lastly,the glucosamine residue at the reducing end of the oligosaccharide were not be enzymatically 6-O-sulfated.Therefore,it can be concluded that each heparin modification enzyme recognize specific sugar residues of non-reducing end from the first GlcA residue at the reducing end of the oligosaccharide substrate and then modify them.Based on this,heparin disaccharide GlcA-GlcNPNZ(2mer-2)was used as the starting sugar receptor to successfully synthesized a unmodified heparin heptasaccharide containing ATⅢ-binding sequence within 11 reaction steps and 8 purification steps with a total yield of 60.24%.The resulting heptasaccharide was determined for its molecular weight by ESI-MS and its structure characterization by NMR.3.Study on the preliminary anticoagulant activity of unmodified heparin heptasaccharideAccording to the chromogenic substrate assay,The semi-inhibitory concentration(IC50)values of anti-FXa activities for unmodified heparin heptaose and fondaparinux were 19.68 ng/mL(9.5 nmol/L)and 14.19 ng/mL(8.2 nmol/L),respectively.Their molar IC50 values are equivalent,suggesting that unmodified heparin heptasaccharide,similar to fondaparinux,exerted specific anti-Xa activity by a single AT-binding sequence in its structure.In a word,heparinase Ⅲ cleavage and mild chemical modification was used establish expanded the chemoenzymatic strategy for heparin synthesis.The synthetic unmodified heparin heptasaccharide containing AT-binding sequence showed comparable specific anti-Xa activity to fonaparinux,and needed less reaction steps.So it is expected to be developed into a lower-cost fondaparinux-like anticoagulant.